1. Field of the Invention
This invention relates to a method of high-sensitive analysis of bile acid, especially bile acid in living body samples.
2. Description of the Background Art
Analysis of bile acid in samples taken from living bodies, such as serum and the like, is important for clinical diagnosis of liver functions as well as for elucidating the cause of such diseases as jaundice, cholelithiasis, and the like.
Conventionally, various methods have been reported for the analysis of bile acid containing cholic acid, deoxycholic acid, chenodeoxycholic acid, and the like. One of the methods recently used for clinical diagnosis involves the use of 3.alpha.-hydroxysteroid dehydrogenase and, as a coenzyme, either a nicotinamide adenine dinucleotide compound (hereinafter referred to as NAD) or a nicotinamide adenine dinucleotide phosphate compound (hereinafter referred to as NADP). Reduced NAD (hereinafter referred to as NADH) or reduced NADP (hereinafter referred to as NADPH) produced in an amount in proportion to the amount of bile acid is then quantitatively measured (Japanese Patent Publication No. 13197/1984). The method, however, is liable to be influenced by bilirubin, and thus requires separation of bile acid in advance. This, in turn, requires a large amount of samples for the analysis. Another drawback of this method is in its low sensitivity. The use of high-sensitivity coloring agents has been proposed in order to overcome this drawback [Proceedings of the 33th of Annual Meeting of Japan Society of Clinical Pathology, 123 (1986); ibid., the 34th Annual Meeting, 124 (1987)]. This method, however, does not bring about a complete solution to the problem.
Another method comprises converting 3-oxosteroid produced into 3-oxo-.DELTA.4-steroid by the action of 3-oxo-.DELTA.4-steroid dehydrogenase for producing formazan twice as much as an amount in conventional method for colorimetric determination. A kit for carrying out this method is commercially available. This method, however, brings about no more effects than making the sensitivity twice as much as that obtained from conventional method.
Japanese Patent Publication No. 36758/1988 discloses a method for the analysis of bile acid by means of an enzymatic cycling reaction of NADH or NADPH which is produced in an amount proportionate to the amount of bile acid. In order to increase the amount of NADH or NADPH by an enzymatic cycling reaction for obtaining a better sensitivity in the analysis, this method requires a step of eliminating surplus NAD or NADP by decomposition with heating in an alkali. Since this involves a complicated procedure, the method is disadvantageous when applied to clinical investigation purposes which require analysis of a large number of samples.
In view of this situation, the present inventors have undertaken extensive studies about the mechanism of the reversible reaction in which oxobile acid is produced using bile acid as a substrate. As a result, the inventors discovered that steroid dehydrogenases derived from normal microorganisms reacted with NADs as conenzymes but hardly reacted with NADPs, and further that a commercially available 3.alpha.-hydroxysteroid dehydrogenase [produced by Toyo Jozo Co., Ltd. "Enzyme Catalog" No. T-27, page 53)] derived from Pseudomonas sp. B-0831 (FERM BP-2376) belonging to the genus Pseudomonas reacted with both NAD and NADP compounds as coenzymes.
Furthermore, the present inventors have found that when a reversible cycling reaction was carried out in a reaction system in which oxobile acid is produced from bile acid using the above steroid dehydrogenase, and an NAD compound or an NADP compound, respectively, as a coenzyme, in the presence of an NADPH compound or an NADH compound, respectively, the amount of NADH or NADPH produced linearly increased over time and its rate of increase was proportionate to the amount of bile acid.
These findings have led to the completion of the present invention.